Deciphering the action of indole analogues on selected targets for neuroprotection in Alzheimer’s disease. Network pharmacology, in silico, and in vitro approaches

Abstract

Alzheimer’s disease is the leading cause of cognitive decline worldwide, and the lack of truly effective disease-modifying treatments has increased interest in phytochemical-derived molecules as alternative therapeutic options for AD. This study combined network pharmacology, molecular docking, molecular dynamics simulations, and in vitro experimental validation to evaluate a series of indole-based analogs as potential candidates for treating Alzheimer’s disease. Protein–protein interaction networks centered on Alzheimer’s-related targets were constructed using STRING, Cytoscape 3.10.2, and DAVID, while Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses helped identify the key biological processes, cellular components, and molecular functions involved in disease progression. The binding affinities between the indole analogs and target proteins were assessed through molecular docking using MolSoft ICM Pro, which also handled protein and ligand preparation, complemented by predictions of ADME/T properties and blood–brain barrier permeability. The most effective compounds were subjected to molecular dynamics simulations using CHARMM-GUI and NAMD software to evaluate the stability of each protein–ligand complex over time. For biological validation, five indole-based analogs were tested at multiple concentrations in N2a neuroblastoma cells using MTT assays alongside a hypoxia cell model to determine IC₅₀ values. Among the tested compounds, A13 emerged as the primary lead compound, demonstrating favorable molecular docking, stable molecular dynamics behavior, negative molecular mechanics/generalized born surface area (MM/GBSA) binding free energy, acceptable ADMET properties, predicted blood–brain barrier permeability, and significant neuroprotective activity. When challenged with glutamate-induced toxicity, A13 and A33 returned IC₅₀ values of 1.87 and 2.28 μM, respectively. Western blot analysis verified that both compounds promoted GSK3β phosphorylation, indicating a meaningful therapeutic mechanism. By combining computational and experimental strategies, this study identified A13 as potential GSK3β inhibitors, supported by robust structural and cell viability evidence that warrants their continued development as therapeutic candidates for Alzheimer’s disease.
Keywords: Alzheimer’s disease, indole analogs, network pharmacology, molecular docking, molecular dynamics simulation